Abstract In response to the National Institutes of Health (NIH), -Center for the Advancement of Science in Space (CASIS), -Request for Application (RFA), -Targeted Research (TR), 18-001 we propose to apply the Organ-Chip technology of Emulate Inc., to assess the effects of space flight in human organs in vitro. Emulate is a newly founded start-up based on technology developed at the Wyss Institute at Harvard University in Cambridge, Massachusetts. The proposed work focuses on adding live imaging functionality to the automated hardware for space already being developed with Implementation Partners SpaceTango through the grant awarded from RFA-TR-16-019. This system will enable experiments in human, in vivo relevant microphysiological systems for understanding of the impact of microgravity and other space flight-imposed stressors on human physiology, disease development and response to interventions. We plan to further demonstrate the value of this automated platform by applying it to study a human innervated Intestine-Chip (hiIC) which includes colonic epithelial cells, lamina propria derived resident immune cells, enteric sensory neurons, and microbiome. Studies will investigate the immune response of this novel model to pathogenic bacteria with and without the incorporation of probiotic bacteria. Studies on identical hardware will be conducted on earth and on the International Space Station, and provide new insights into the influence of the space environment on immune response. The new imaging functionality will allow for real-time imaging of the organ-chips throughout their space journey and infection with pathogenic bacteria, giving an unprecedented view of how the system responds in this unique environment. Successful implementation of our space compatible hardware, and extending its application to our novel hiIC-chip, will show the value of an in vivo relevant, in vitro platform that can be utilized by the scientific community for the evaluation of the impact of microgravity in physiology and disease of a number of human organs, and support drug development in novel, clinically relevant ways.